Assessment of reproductive and developmental effects of graphene oxide on Japanese medaka (Oryzias latipes)

Highlights

• Single IP-injected graphene oxide (GO) tended to interrupt medaka breeding.

• Agglomerated GO nanoparticles were observed in medaka gonads 21days post-injections.

• Histopathological alterations of medaka gonads by GO are found to be minimum.

• Morphology of granulosa and Leydig cells in gonads remained unaltered by GO.

• No endocrine disrupting effects of GO were observed in liver and kidneys of medaka.

Abstract

Due to its unique properties, graphene oxide (GO) has potential for biomedical and electronic applications, however environmental contamination including aquatic ecosystem is inevitable. Moreover, potential risks of GO in aquatic life are inadequately explored. Present study was designed to evaluate GO as an endocrine disrupting chemical (EDC) using the model Japanese medaka (Oryzias latipes). GO was injected intraperitoneally (25–200 μg/g) once to breeding pairs and continued pair breeding an additional 21 days. Eggs laid were analyzed for fecundity and the fertilized eggs were evaluated for developmental abnormalities including hatching. Histopathological evaluation of gonads, liver, and kidneys was made 21 days post-injection. LD₅₀ was found to be sex-dependent. Fecundity tended to reduce in a dose-dependent manner during early post-injection days; however, the overall evaluation showed no significant difference. The hatchability of embryos was reduced significantly in the 200 μg/g group; edema (yolk and cardiovascular) and embryo-mortality remained unaltered. Histopathological assessment identified black particles, probably agglomerated GO, in the gonads of GO-treated fish. However, folliculogenesis in stromal compartments of ovary and the composition of germinal elements in testis remained almost unaltered. Moreover, granulosa and Leydig cells morphology did not indicate any significant EDC-related effects. Although liver and kidney histopathology did not show GO as an EDC, some GO-treated fish accumulated proteinaceous fluid in hepatic vessels and induced hyperplasia in interstitial lymphoid cells (HIL) located in kidneys. GO agglomerated in medaka gonads after 21-days post-injection. However, gonad histopathology including granulosa and Leydig cells alterations were associated with GO toxicity rather than EDC effects.

Introduction

In recent years, graphene, a sp²-bonded carbon nanosheet, has been a promising biomaterial for diagnostic and therapeutic applications. The graphene family of nanomaterials including few-layer-graphene, ultrathin graphite, GO, reduced graphene oxide (rGO), graphene nanoplatelets, graphene nanosheets and others, varied in layer numbers, layer dimensions, surface chemistry, density or purity (Novoselov et al., 2012; Bianco, 2013; James and Tour, 2013). Because of their high water solubility (6.6 μg/mL in de-ionized water; Konios et al., 2014; Johnson et al., 2015), and use as precursors of chemically generating large-scale graphene structures, GO nanosheets have the potential to become a biomaterial for application both in vivo and in vitro models. As a graphene derivative, GO has a large two-dimensional plane, which provide ultrahigh, specific surface area to load drugs through surface adsorption, π-π interaction, hydrogen bonding, and others. GO is biocompatible and nontoxic which makes it a promising candidate for construction of drug carriers. However, several published reports indicate that GO is able to produce toxic effects in various animal models (reviewed by Ema et al., 2016) including fish (reviewed by Dasmahapatra et al., 2019). Moreover, even though GO could yield stable suspensions in water initially after being exfoliated into monolayer sheets, it may aggregate immediately in biomedia (blood or tissue fluids), especially after loading with anticancer drugs. Hence there is a need to investigate the toxic effects of GO considering all possible ways including various exposure routes, and to ensure that the methods used for its application have no toxic impact on animals (Kurantowicz et al., 2015).

Aquatic environments are the ultimate sink for many contaminants, either due to direct discharge or by hydrological and atmospheric processes. In recent years, increasing amount of carbon-based nanomaterials (CNM) have been observed in aquatic systems. However, the concentration of CNM in the aquatic environment is very low (ng/L), even lower than the lowest observed effect concentrations (LOEC) reported on different aquatic organisms (Freixa et al., 2018). Moreover, CNM interact with other micro-pollutants, thereby modifying the original toxicity of contaminants. Since fish occur in virtually all aquatic environments and play a major role in aquatic food webs, they are useful models for monitoring pollutants in aquatic environment. Small laboratory fish models, including zebrafish (Danio rerio) and Japanese medaka (Oryzias latipes), are widely used as experimental models for monitoring pollutants in the environment.

The toxicity of GO in fish, especially in zebrafish (Danio rerio) was found to be concentration-dependent (Bangeppagari et al., 2019; Dasmahapatra et al., 2019; Yang et al., 2019), and did not affect embryonic development at lower concentrations. However, higher concentration showed significant impact on embryo mortality, hatching, and disorder in cardiovasculature (Bangeppagari et al., 2019). Exposure of zebrafish embryos to GO for five consecutive days did not induce acute developmental toxicity, however, hatching and locomotor behavior were altered (Yang et al., 2019). Moreover, genes related to nervous and immune systems were upregulated (Yang et al., 2019). Proteomic analysis in zebrafish larvae identified down-regulation of several oxidative stress-related proteins by GO (Zou et al., 2018; Dasmahapatra et al., 2019). In tilapia fish, oral exposure of GO did not alter behavior, body weight, and the intestinal bacterial population after 30 days of treatment. However, the expression of several oxidative stress-related enzymes was down regulated significantly in liver, but remained unaltered in spleen, gill, intestine, and muscle (Ma et al., 2016). In Geophagus iporangensis, GO exposure decreased metabolic rate compared to control fish (Medeiros et al., 2019). Several oxidative stress-related enzymes were increased in the climbing perch (Anabas testudineus) after 24 h of single intraperitoneal (ip) injection of GO (Paital et al., 2019).

Although in fish models, evaluation of GO was mainly focused on toxicological endpoints mediated probably by oxidative stress-related enzymes and pathways, the role played by GO as an EDC needs to be investigated. A recent report has indicated the potential of GO as a thyroid endocrine disruptor in tadpoles of Xenopus laveis (Li et al., 2019). Due to limited data, the effects of different nanoparticles on endocrine system need to be interpreted carefully (Iavicoli et al., 2013). Previously, our laboratory evaluated the toxic potential of GO administered orally to Sprague-Dawley rats using liver and kidney tissues. We observed that oral administration of higher doses of GO (20–40 mg/kg, five consecutive days) induced cellular damages in liver and kidneys as well as enhanced enzyme activities related to oxidative stress (Patlolla et al., 2016, 2017).

The aim of the present study was to assess the toxic potential of GO using Japanese medaka fish as an aquatic animal model. In addition, we also evaluated on the endocrine disrupting effects of GO targeting testis and ovary. Leydig cells in the testis produced male hormones (testosterone) while the perifollicular cells, especially theca and granulosa cells, in the ovary of medaka are responsible for female hormones (estrogen). This fish species is a recognized model to evaluate EDCs in the environment and several Test Guidelines (TG) are included in publications made by Organization for Economic Cooperation and Development (OECD, 2018). We used sexually mature, reproductively active male and female intact Japanese medaka fish maintained in glass aquaria under standard laboratory conditions (25±1 °C; 16L: 8D light cycle). We focused on fecundity and histopathology of gonads (testis and ovary) as toxicological and reproductive (endocrine) endpoints 21 days after single ip injection of GO (25–200 μg/g). In fish, fecundity can be characterized by a wide spectrum of disorders including endogenous hormone levels, activities of gonads, as well as the behavior of the fish during mating and external fertilization. However, fecundity in medaka is highly variable and reproducibility is questionable (Hutchinson et al., 2006) even though high concentrations of EDCs such as 17 alpha-ethinylestradiol (EE2) or 17 beta-trenbolone (TB) significantly reduced fecundity (Park et al., 2009). Moreover, in addition to histopathology of gonads, and because of the significant role played by liver and kidneys in fish reproduction (OECD, 2010, 2018), we conducted histopathological evaluation of these two organs focusing on EDC effects. To our knowledge, the toxicity and EDC effects of GO in reproductively active Japanese medaka adults remained unexplored.